The goal of this research project proposal is to co-opt dysfunctional innate immune signaling in KRAS- driven lung cancers as a therapeutic vulnerability. Specifically, my laboratory has focused for many years on targeting TBK1 to inhibit the production of cytokines and chemokines such as IL-6 and CCL5 that are pro- tumorigenic and immune suppressive. For example, TBK1 inhibition sensitizes tumors to MEK inhibition, as well as PD-1 blockade. Over the past several years it has also become increasingly apparent that viral sensing pathways, such as RIG-I/MAVS or cGAS/STING play a key role in re-directing TBK1 to activate IRF3/STAT1 and initiate a cytotoxic anti-viral response. How KRAS mutant lung cancers, especially those with STK11/LKB1 co-mutation, avoid this response and preferentially activate NF-?B/STAT3 survival signaling has remained unclear. While developing triple combination therapies to inhibit TBK1/MEK signaling and suppress adaptive transcriptional feedback, we made the serendipitous observation that KRAS-LKB1 (KL) mutant lung cancers epigenetically silence STING. Detailed studies in KL cells unveiled a mechanistic connection between enhanced DNMT1 activity and the need to avoid detection of mitochondrial DNA, which accumulates due to damaged mitochondria. Re-activating STING expression in KL cells results in cellular cytotoxicity and enhanced immunogenicity, especially when combined with STING agonism. Thus, instead of inhibiting multiple signaling pathways downstream KRAS, these studies uncover a straightforward vulnerability that could be more readily co-opted therapeutically. The broad, long term objective of this proposal is therefore to characterize the epigenetic mechanism of STING silencing and to co-opt this state into a tumor vulnerability. Forcing cells to deal with the consequences of STING re-expression, while driving its activity via DNA damage or direct STING agonism, has important therapeutic potential for this major subset of KRAS-driven lung cancer. Given the unclear therapeutic window of targeting three or more KRAS downstream pathways, which is required for long-term durable response in animal models, this simpler strategy, which can also re-engage anti-tumor immunity, has significant potential. Moreover, in addition to the in vivo studies we propose, our novel model system using patient-derived xenograft and direct patient-derived organotypic tumor spheroids (XDOTS and PDOTS) provides rapid validation in actual explanted tumors. Specific aims are to: 1) Optimize strategies to reverse epigenetic silencing of STING in KL tumors, 2) Develop combination therapy strategies with specific agents that promote mitotic slippage, and 3) Utilize immune-competent models to explore the direct role of STING priming on adaptive T cell responses. Through these complementary studies, the goal is to rewire the cytokine circuitry of KRAS-driven lung cancer to engage this cytotoxic anti-viral signaling machinery and ultimately to overcome intrinsic resistance to immune checkpoint inhibitor therapy.